Abstract

The objective of this study was to determine the effects of different doses
of three natural essential oils(EOs) from semi-arid medicinal
plants (Cotula cinerea, Juniperus phoenicea, and Artemisia
campestris) on in vitro camel rumen fermentation of
Atriplex halimus, a plantthat is eaten by the dromedary. The
levels added were 0.0, 0.10, 0.25 and 1.0 g/liter of the fermentation
medium.

All the oils decreased the production of gas and methane per unit of
substrate DM incubated. N-NH3 concentration was reduced by the
oil from Juniperus phoenicea.

Key words: ammonia, dromedary, essential oils, methane

Introduction

In recent studies, a variety of compounds and substances have been evaluated
for their ability to reduce methane emissions from rumen fermentations
in vitro and in vivo (Beauchemin et al 2008;
Bunglavan et al 2010; Buddle et al 2011; Patra et al 2012;
Adelusi et al 2015; Vongkhamchanh et al 2015; Sengsouly and Preston 2016).
The inclusion of essential oils of medicinal plants, as natural
alternatives, has been investigated for inclusion in ruminant diets for
improving feed efficiency, as well as decreaet alsing the adverse envirental
effects of ruminants (Wallace 2004).

The aim of the present study was to investigate the effect of different
levels of essential oils on methane pproduction fromCotula cinerea,
Juniperus phoenicea, Artemisia campestris in anin vitro
camel rumen fermentation.

Materials and methods

Forage Samples

Sampling of plant material was done in a salty area named Benguecha (Taleb
El Oued Province-Algeria). The plant Atriplex halimus
(Amaranthaceae) was selected as the feed substrate for incubation. It was
chosen as being the main component of the herbaceous layer along the route
taken by camels. A preference by camels for this plant is well known; also
that it is effectively degraded by the dromedary (Medila 2015).

Plant essential oil

Semi-arid native medicinal plants (Cotula cinerea, Juniperus phoenicea,
Artemisia campestris) were collected from the El Oued region of Algeria.
The essential oils from these plants were obtained through steam distillation
using the “Celevenger” procedure (Clevenger, 1928). The distillation was carried
out for four hours in accordance with the recommendations of the European
Pharmacopoeia (AFNOR 2000). The extractions were repeated five times to confirm
the return earned by the mode used. The essential oil was stored and maintained
optimally at 4 °C protected from light.

Treatments and design

Three different doses were taken in the case of each essential oil: 0.10,
0.25 and 1.0 g/liter of in vitro fermentation medium. Each
treatment was incubated in triplicate.

In vitro gas production

The in vitro fermentation was conducted according to the technique
of calibrated glass syringes (100ml capacity) described by Menke and
Steingass (1988). 200 mg dry weight of the samples were placed in triplicate
in 100 ml calibrated glass syringes. Rumen liquid was collected from 3
dromedaries slaughtered in the morning. These animals were randomly
selected, of different age and sex. The rumen liquor was bubbled with C0
2 for about 2 minutes and filtered through 4 layers of muslin cloth.
The strained rumen liquor was mixed with a phosphate-bicarbonate buffer
(Menke et al 1979) in a 2:1 ratio and 40ml of this mixture were introduced
in each syringe for incubation. The level of the piston was recorded and the
syringes were placed in an incubator (39 ± 0.5°C). Gas volumes were recorded
after 24 h of incubation.

At the end of the incubation, the total gas produced in each fermenter was
collected in gas bags and quantified by displacement of water. Samples were
drawn from the total gas produced and fractioned for methane and carbon
dioxide by the method of Fievez et al (2005). A 2ml sample was taken from
the gas bag with the help of syringe and needle. It was then injected
through the nozzle of another syringe containing 2 ml of 10M NaOH. The
displacement of gas caused by the 10M NaOH indicated the volume of methane.

N-NH3 was determined by the colorimetric technique of Chaney and
Marbach (1962).

Statistical analysis

The data were analyzed using SAS software (SAS 2013). The effects of
treatments were compared with those of the control using the Dunnett test.
Significant differences were declared at P < 0.05.

Results

Relative to the control, all three sources of essential oils reduced
production of methane and total gas (Table 1; Figures 1-3). Rumen ammonia
levels were reduced by oil from Juniperus phoenicea but were not
affected by essential oils from Cotula cinereal and
Artemisia campestris.

Table 1. Mean values for gas and methane production, and N-NH3 concentration, in an in vitro rumen microbial fermentation of three plant essential oils

Gas production (ml)

Methane(ml)

N-NH3
(mM)

Control

30.9

15.2

7.4

Cotula cinerea

0.1

30.1

13.6

7.2

0.5

29.6

12.5

7.1

1

29.1

11.1

6.9

SEM

1.22

0.88

0.41

p

0.031

0.009

0.64

Juniperus phoenicea

0.1

30.2

13.4

7.3

0.5

29.2

10.9

5.7

1

29.5

9.2

4.5

SEM

1.16

0.91

0.36

p

0.045

<0.001

0.007

Artemisia campestris

0.1

30.3

13.5

7.4

0.5

29.1

12.9

7.3

1

26.1

11.9

7.1

SEM

1.63

0.74

0.42

p

0.044

<0.001

0.114

Figure 1. Effect of Cotula cinérea essential oil on methane production in an in vitro fermentation of Atriplex halimus

Figure 2. Effect of Juniperus phoenicea essential oil on methane production in an in vitro fermentation of Atriplex halimus

Figure 3. Effect of Artemisia campestris essential oil on methane production in an in vitro fermentation of Atriplex halimus

Discussion

In the present study, essential oil from Juniperus phoenicea was
more potent than oil from Cotula cinérea andArtemisia
campestris in lowering methane and N-NH3
production. The two latter sources of essential oil appeared to reduce
methane production but did not affect N-NH3 concentration. The
decrease in N-NH3 concentration with the addition of
Juniperus phoenicea essential oil was probably consistent with direct
inhibition of proteolysis, and peptidolitic and deamination processes. Patra
(2011) demonstrated that essential oils can reduce NH 3
concentration and protein deamination by inhibiting hyper-ammonia producing
bacteria. McIntosh et al (2003) demonstrated that a commercial blend of
essential oil compounds reduced the rate of amino acid deamination and
inhibited the growth of a specific group of ammonia hyper-producing
bacteria. Addition of thymol (an essential oil) to a medium containing rumen
liquid resulted in an accumulation of amino acid nitrogen and a decrease in
the ammonia nitrogen according to Castillejos et al (2007).

Many studies have documented reduction in methane production by essential
oils (Agarwal et al 2009, Patra et al 2012, Jahani-Azizabadi 2014, Joch et
al 2017). Oils from eucalyptus, garlic, origanum and peppermint
supplementations all educed methane production in the range of 18 to 87.0%
according to Parra and Yu (2012). Knapp et al (2014) indicated that the
effect of essential oils in reducing in vitro CH4
production was through a direct inhibition of methanogenic archaea and/or an
indirect depression of some microbial metabolic processes involved in
methanogenesis. However, Beauchemin and McGinn (2006) did not reveal any
effect on methanogenesis in a study done in vivo.

The practical application of the findings reported in this paper have yet to
be demonstrated.

Menke K H, Raab L, Salewski A, Steingass H., Fritz D and Schneider W
1979 The estimation of the digestibility and metabolizable energy content
of ruminant feedingstuffs from the gas production when they are incubated with
rumen liquor in vitro. Journal of Agricultural Science 93: 217–222.

Menke K H and Steingass H 1988
Estimation of the energetic feed value obtained from chemical analysis and
in vitro gas production using rumen fluid. Animal Research and
Development. 28:7-55.

Patra A K and Environ Monit A 2012
Enteric methane mitigation technologies for ruminant livestock: a synthesis
of current research and future directions.
Environmental Monitoring and Assessment.184(4):1929-52.

Patra A K and Yu Z 2012
Effects of Essential Oils on Methane Production and Fermentation by, and
Abundance and Diversity of, Rumen Microbial Populations. Applied and
Environmental Microbiology. 78(12):4271-4280.

Sengsouly P and Preston T R 2016
Effect of rice-wine distillers’ byproduct, biochar and sweet or bitter
cassava leaves on gas production in an in vitro incubation using
ensiled cassava root as substrate. Livestock Research for Rural Development.
Volume 28, Article #190. Retrieved October 15, 2017, from
http://www.lrrd.org/lrrd28/10/seng28190.html

Vongkhamchanh B, Inthapanya S and Preston T R 2015
Methane production in an in vitro rumen fermentation is reduced when the
carbohydrate substrate is fresh rather than ensiled or dried cassava root,
and when biochar is added to the substrate. Livestock Research for Rural
Development. Volume 27, Article #208.
http://www.lrrd.org/lrrd27/10/bobb27208.html